Method of increasing expression of nucleic acid molecules in plants using multiple transcription units

ABSTRACT

A method of increasing expression of a product encoded by a nucleic acid molecule is provided where in one embodiment multiple plant transcription units comprising a promoter and the nucleic acid molecule are provided. In another embodiment multiple plant transcription units are provided where each promoter is different from the other. In an embodiment the multiple plant transcription units may comprise two, three, four or more plant transcription units. In another embodiment the promoter may be selected from an embryo promoter. Another embodiment provides the promoter may be a globulin promoter. A further embodiment provides the product encoded may be selected from hepatitis b, aprotinin, or a cellulase. Still further embodiments provide the product may be selected from the cellulases E1 and CBH1.

REFERENCE TO RELATED APPLICATION

This application claims priority to previously filed and co-pending application U.S. Ser. No. 61/549,343, filed Oct. 20, 2011, and is a continuation-in-part of previously filed and copending application U.S. Ser. No. 13/558,834 filed Jul. 26, 2012, which claims priority to U.S. Ser. No. 61/512,347 filed Jul. 27, 2011, the contents of each are incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under contract No. DOE DE FG36 GO88025 awarded by the United States Department of Energy. The Government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 15, 2012, is named AB00016.txt and is 46,682 bytes in size.

BACKGROUND

Plants are a promising system for synthesis of foreign proteins due to their potential for low-cost high-volume production, ability to perform post-translational modifications, and lack of possible pathogen contamination. Many plant systems ranging from dicot model species such as tobacco and Arabidopsis to monocots including rice and maize have been used to express genes for disease resistance, enhanced nutrient quality, and production of pharmaceuticals and industrially valuable proteins (reviewed in Streatfield, 2007, Khan, 2010, Khan, in press). There has been a continued effort to increase and tightly regulate expression of proteins and products in plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence of the approximately 1.4 kb globulin-1 promoter (SEQ ID NO: 1) with the regulatory region at bases 1-1386 (SEQ ID NO: 2), the TATA box at bases 1354-1360 and the untranslated region at bases 1387-1401 shown in italics (SEQ ID NO: 3). An extra 43 bases appears in bold and below the sequence (SEQ ID NO: 4).

FIG. 2 shows the nucleotide sequence of the approximately 3 kb extended globulin-1 promoter (SEQ ID NO: 5) with the untranslated region in bold (SEQ ID NO: 6) and the translation start codon capitalized.

FIG. 3 shows the nucleotide sequence of the globulin-2 promoter (SEQ ID NO: 8) with the untranslated leader sequence in bold (SEQ ID NO: 9) and the translation start codon capitalized.

FIG. 4 shows the nucleotide sequences of the pr26 promoter (SEQ ID NO: 11) with the predicted untranslated leader in bold (SEQ ID NO: 12).

FIG. 5 shows the nucleotide sequence of the pr36 promoter (SEQ ID NO: 14) with the putative TATA box underlined and the ATG in bold.

FIGS. 6A and 6B are graphic representations of constructs. Various combinations of (a) the E1 or (b) CBHI coding region under control of different promoters were prepared for expression in the maize embryo. Black, white, striped, or stippled blocks represents the globulin-1 (Glb1), the globulin-2 (Glb2), the pr26, or pr36 promoters, respectively. The gray blocks represent either the E1 or CBHI coding regions.

FIGS. 7A and B are a graphs showing cellulase expression in T₁ positive seed. The mean RFU (relative fluorescence units) for each construct is shown, either for all seed (black) or the highest ten seed (gray).

FIGS. 8A and B shows two gels of Western blots, FIG. 8A showing E1 quantitation and

FIG. 8B showing CBH1 quantitation.

FIG. 9A shows a gel of Southern blot analysis, FIG. 9B shows DNA blot analysis of constructs.

FIGS. 10A and 10B are graphs showing expression in pooled T₂ seed compared to the average expression in T₁ seed for the same event, with 10A showing results for constructs CEK and CEB and 10B showing results from constructs CCF and CCG.

FIG. 11 shows the sequence of the optimized hepatitis B surface antigen nucleotide sequence (SEQ ID NO: 16) used in the experiments along with the Barley Alpha Amylase Signal Sequence (BAASS) (SEQ ID NO: 17) which is in italics with the ATG start codon and stop codon sites in bold.

FIG. 12 is a plasmid map of the vector used to express hepatitis B surface antigen.

FIG. 13 is a plasmid map of the vector used to express aprotinin.

FIG. 14 shows the E1 sequence (SEQ ID NO: 18) used in the experiments.

FIG. 15 shows the CBHI sequence (SEQ ID NO: 19) used in the experiments.

DESCRIPTION

In order to express a nucleic acid molecule in a plant cell, a plant transcription unit (PTU) is provided which comprises at least a promoter and the nucleic acid molecule. When referring to a plant transcription unit is meant a promoter operably linked to a nucleic acid molecule and which can be introduced into a plant cell. The nucleic acid molecule (also referred to here as a “gene of interest”) transcribes RNA to express a product. The promoter drives expression of the nucleic acid molecule and the product encoded by the nucleic acid molecule accumulates in the plant cell. In one embodiment the nucleic acid molecule expresses a polypeptide in the plant cell. The PTU may optionally include other PTU components, in addition to the promoter which drives the nucleic acid molecule of interest and the nucleic acid molecule of interest. These components may include any component of the vector (other than and in addition to the promoter driving the nucleic acid molecule of interest and the nucleic acid molecule of interest) useful in producing or enhancing production of the product encoded by the nucleic acid molecule. By the way of example, without limitation, such components may include an untranslated leader, a polyadenylation sequence, a signal sequence, an enhancer sequence, a selectable marker or the like. Examples are discussed further below. It will be evident to one skilled in the art many such non-promoter components may be used in a vector, depending upon the particular application.

Rather than suppressing expression, the inventors have found that use of multiple PTUs results in increased accumulation of product in the plant cells and in a plant compared to expression of a product and accumulation using one PTU. Here described in one embodiment is the use of multiple PTUs having the same promoter and same nucleic acid molecule expressing the product. Another embodiment is to a method using multiple PTUs where each PTU has a different promoter. The multiple PTUs may include at least two PTUs and in another embodiment may include two, three, four or more PTUs. In an embodiment, the other components may be the same for each PTU. This is particularly useful for reliable production of product from the nucleic acid molecule with multiple PTUs having different promoters.

The product may be any product encoded by the nucleic acid molecule. By way of example, in one embodiment the product expressed by the nucleic acid molecule may be selected from hepatitis B or aprotinin or a cellulase enzyme. The method is particularly useful when the product expressed is a cellulase enzyme, and in another embodiment may be endo-β-1,4-glucanase (E1) or exo-β-1,4-glucanase (CBH1). A still further embodiment provides that the PTU comprises a promoter comprising an embryo preferred promoter. One example provides the promoter is a globulin promoter. By way of example without limitation the promoter may be selected from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 20 or SEQ ID NO: 21.

Any plant-compatible promoter may be used in the methods disclosed. By “promoter” is meant a regulatory region of DNA capable of regulating the transcription of a sequence linked thereto. It usually comprises a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular coding sequence. The promoter is the minimal sequence sufficient to direct transcription in a desired manner. The term “regulatory region” is also used to refer to the sequence capable of initiating transcription in a desired manner. A promoter can additionally comprise other recognition sequences generally positioned upstream or 5′ to the TATA box, referred to as upstream promoter elements, which influence the transcription initiation rate. It is recognized that having identified the nucleotide sequences for the promoter regions disclosed herein, it is within the state of the art to isolate and identify further regulatory elements in the 5′ region upstream from a promoter including any particular promoter region. Thus the promoter regions are generally further defined by comprising upstream regulatory elements such as those responsible for tissue and temporal expression of the coding sequence, enhancers and the like. In the same manner, the promoter elements which enable expression in the desired tissue such as the embryo can be identified, isolated, and used with other core promoters to confirm embryo-preferred expression. By core promoter is meant the sequence sometimes referred to as the TATA box (or similar sequence) which is common to promoters in all genes encoding proteins. Thus the upstream promoter can optionally be used in conjunction with its own or core promoters from other sources. The regulatory regions referred to herein may include the sequence which initiates transcription in the desired manner and may be linked with a TATA box or where an untranslated leader is used, may use an untranslated leader from another nucleic acid molecule, in addition to using the sequence natively associated with the regulatory region.

Various plant promoters are available to those skilled in the art. These can be plant gene promoters, such as, for example, the ubiquitin promoter (European patent application no. 0 342 926); the promoter for the small subunit of ribulose-1,5-bis-phosphate carboxylase (ssRUBISCO) (Coruzzi et al., 1984; Broglie et al., 1984); or promoters from the tumor-inducing plasmids from Agrobacterium tumefaciens, such as the nopaline synthase, octopine synthase and mannopine synthase promoters (Velten and Schell, 1985) that have plant activity; or viral promoters such as the cauliflower mosaic virus (CaMV) 19S and 35S promoters (Guilley et al., 1982; Odell et al., 1985), the figwort mosaic virus FLt promoter (Maiti et al., 1997) or the coat protein promoter of TMV (Grdzelishvili et al., 2000). Alternatively, plant promoters such as heat shock promoters for example soybean hsp 17.5-E (Gurley et al., 1986); or inducible promoters such as ethanol-inducible promoters (Caddick et al., 1998) may be used. Exemplary inducible promoters include ecdysone receptor promoters, U.S. Pat. No. 6,504,082; promoters from the ACE1 system which responds to copper (Mett et al. PNAS 90: 4567-4571 (1993)); In2-1 and In2-2 gene from maize which respond to benzenesulfonamide herbicide safeners (U.S. Pat. No. 5,364,780; Hershey et al., Mol. Gen. Genetics 227: 229-237 (1991) and Gatz et al., Mol. Gen. Genetics 243: 32-38 (1994)) Tet repressor from Tn10 (Gatz et al., Mol. Gen. Genet. 227: 229-237 (1991); or from a steroid hormone gene, the transcriptional activity of which is induced by a glucocorticosteroid hormone. Schena et al., Proc. Natl. Acad. Sci. U.S.A. 88: 10421 (1991); the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides; and the tobacco PR-1a promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J. 14(2):247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156). See International Patent Application No. WO 91/19806 for a review of well-known plant promoters.

Tissue-preferred promoters can be utilized to target enhanced transcription and/or expression within a particular plant tissue. When referring to preferential expression, what is meant is expression at a higher level in the particular plant tissue than in other plant tissue. Examples of these type of promoters include seed preferred expression such as that provided by the phaseolin promoter (Bustos et al. (1989) The Plant Cell Vol. 1, 839-853), and the maize globulin-1 gene, Belanger, et al. (1991) Genetics 129:863-972. For dicots, seed-preferred promoters include, but are not limited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-preferred promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, γ-zein, waxy, shrunken 1, shrunken 2, globulin 1, an Ltp1 (See, for example, U.S. Pat. No. 7,550,579), an Ltp2 (Opsahl-Sorteberg, H-G. et al., (2004) Gene 341:49-58 and oleosin genes. See also WO 00/12733, where seed-preferred promoters from end1 and end2 genes are disclosed. Seed-preferred promoters also include those promoters that direct gene expression predominantly to specific tissues within the seed such as, for example, the endosperm-preferred promoter of γ-zein, the cryptic promoter from tobacco (Fobert et al. (1994) “T-DNA tagging of a seed coat-specific cryptic promoter in tobacco” Plant J. 4: 567-577), the P-gene promoter from corn (Chopra et al. (1996) “Alleles of the maize P gene with distinct tissue specificities encode Myb-homologous proteins with C-terminal replacements” Plant Cell 7:1149-1158, Erratum in Plant Cell 1997, 1:109), the globulin-1 promoter from corn (Belanger and Kriz (1991) “Molecular basis for Allelic Polymorphism of the maize Globulin-1 gene” Genetics 129: 863-972 and GenBank accession No. L22344), promoters that direct expression to the seed coat or hull of corn kernels, for example the pericarp-specific glutamine synthetase promoter (Muhitch et al., (2002) “Isolation of a Promoter Sequence From the Glutamine Synthetase₁₋₂ Gene Capable of Conferring Tissue-Specific Gene Expression in Transgenic Maize” Plant Science 163:865-872 and GenBank accession number AF359511) and to the embryo (germ) such as that disclosed at U.S. Pat. No. 7,169,967.

In one embodiment the promoter used in the multiple PTUs may be an embryo preferred promoter. When referring to an embryo preferred promoter is meant that it expresses an operably linked sequence to a higher degree in embryo tissue that in other plant tissue. It may express during embryo development, along with expression at other stages, may express strongly during embryo development and to a much lesser degree at other times.

Globulin promoters are known to one skilled in the art. Such promoters are obtained from nucleic acid molecules which encode a globulin protein. The two most abundant proteins in maize embryos are saline-soluble, water-insoluble globulins, one being a 63,000 Da molecular weight protein encoded by the globulin-1 gene, the other a 45,000 Da molecular weight protein encoded by the globulin-2 gene. See. E.g, Kriz (1989) Biochem Genet. 27(3-4):238-51 and Belanger, F. C. and Kriz, A. L. (1991) “Molecular basis for allelic polymorphism of the maize globulin-1 gene” Genetics 129, 863-872. Belanger et al. note that the protein is readily detected in a Coomassie-stained gel of protein extracts from embryos and several alleles have been recognized. Belanger et al. (1991), at 865. One example is the promoter associated with the nucleic acid molecule encoding globulin-1. Globulin-1 is the most abundant protein in maize embryos, is a vicilin-like storage protein comprising 10-20% of the maize embryo protein and encoded by the globulin-1 gene. See, e.g., Liu et al. (1992) MNL Vol. 22: 108-109. Where a null allele is present no globulin 1 protein is produced. Belanger et al. (1991). One skilled in the art appreciates that nucleic acid molecules that encode the globulin-1 protein (as well as other products which may be produced by a nucleic acid molecule) are well known and readily identified using techniques available to one skilled in the art and as discussed here, including, by way of example without limitation, comparison to known sequences, preparation of a library and screening with a probe, antibody binding, using Northern, Southern or Western blots, among the many avenues available. Examples, without intending to be limiting, of globulin promoters include the 1.45 kb maize globulin-1 promoter plus untranslated leader described by Belanger and Kriz, 1991, supra and GenBank accession L22344 shown in FIG. 1 and is SEQ ID NO: 1. The 1.4 kb Belanger et al. globulin-1 promoter referred to includes the regulatory region of bases 1-1386 (SEQ ID NO: 2), the TATA box of bases 1354-1360 and the 5′ untranslated region of bases 1387-1401 (shown in italics and which is SEQ ID NO: 3). The version of the promoter used in the experiments here included an extra 43 bases (SEQ ID NO: 4, shown in bold below the promoter in FIG. 1) which do not form a part of the promoter and believed to be a downstream portion on chromosome 1 of the maize gene, which may originate from a retrotransposon.

Another example of a globulin promoter is a nucleotide sequence natively associated with the nucleotide sequence coding for Zea mays extended globulin-1 and in an example comprises SEQ ID NO: 5 shown in FIG. 2. This promoter was first described in U.S. Pat. No. 7,169,967, and is also shown in U.S. Ser. No. 13/558,834, each incorporated herein by reference in its entirety. It includes the proximal approximately 3 kb of a maize extended globulin-1 promoter plus untranslated leader (SEQ ID NO: 5). Transgenic plants generated using this sequence show significantly increased expression over those generated using the Belanger et al. 1.4 kb maize globulin-1 promoter plus untranslated leader described above, which has previously been deployed to express transgenes in maize seeds (Hood et al., 2003; Woodard et al., 2003). The extended globulin-1 promoter plus untranslated leader sequence of patent '967 is highly embryo preferred in its expression pattern, as is the previously cloned globulin-1 promoter sequence of Belanger et al. The untranslated leader sequence (SEQ ID NO: 6) in FIG. 2 is shown in bold type and the translation start codon is capitalized. The regulatory region minus the untranslated leader is SEQ ID NO: 7.

A further example of a globulin promoter is the nucleotide sequence natively associated with the nucleotide sequence coding for globulin-2. See one example described by Wallace and Kriz, “Nucleotide sequence of a cDNA clone corresponding to the maize globulin-2 gene” Plant Physiol. 95, 973-975 (1991) and shown at GenBank Accession No. X53715.1. Another example, is the globulin-2 promoter shown at GenBank Accession No. AR947679 and at U.S. Pat. No. 7,112,723 (shown there as sequence 4 and FIG. 3), incorporated herein by reference in its entirety. FIG. 3 shows the entire 3 kb promoter as SEQ ID NO: 8, with the untranslated leader sequence in bold (SEQ ID NO: 9) and the translation start codon capitalized. The regulatory region minus the untranslated region is SEQ ID NO: 10.

In a still further example of an embryo preferred promoter, a promoter from a maize abscisic acid-inducible nucleic acid molecule having preferential expression in plant embryo tissues may be employed. Such a promoter is described at Streatfield et al. (2010) “Identification of maize embryo-preferred promoters suitable for high-level heterologous protein production” GM Crops 1:1-11, at GenBank EA076965 (referred to there and here as pr26) and U.S. Pat. No. 7,183,109 (shown there at FIG. 3 and as sequence 3), incorporated herein by reference in its entirety. The pr26 promoter is shown in FIG. 4 and is SEQ ID NO: 11. The predicted minimal extent of the untranslated leader is shown in FIG. 4 in bold (SEQ ID NO: 12) and the start codon capitalized. The regulatory region minus the untranslated region is SEQ ID NO: 13.

Yet another example of an embryo preferred promoter is the promoter shown at Streatfield et al. (2010), supra, at GenBank HM635908.1 (referred to there and here as pr36) and at US patent Publication 20110091976 (at there at FIG. 1 and as sequence 2) incorporated herein by reference in its entirety. The pr36 promoter is shown in FIG. 5 and is SEQ ID NO: 14. The putative TATA box is underlined, based on consensus sequences and the ATG is in bold.

Clearly, many variations in use of the promoters which may be used in the methods described are available to one skilled in the art.

The multiple PTUs are used with a nucleic acid molecule encoding the product of interest, also referred to as the gene of interest. The “gene of interest” refers to a nucleotide sequence that encodes for a desired polypeptide or protein but also may refer to nucleotide sequences that do not constitute an entire gene, and which do not necessarily encode a polypeptide or protein. For example, when used in a homologous recombination process, the promoter may be placed in a construct with a sequence that targets an area of the chromosome in the plant but may not encode a protein. The promoter can be used to drive mRNA that can be used for a silencing system, such as antisense, and in that instance, no protein is produced. Means of increasing or inhibiting a protein are well known to one skilled in the art and, by way of example, may include, transgenic expression, antisense suppression, co-suppression methods including but not limited to: RNA interference, gene activation or suppression using transcription factors and/or repressors, mutagenesis including transposon tagging, directed and site-specific mutagenesis, chromosome engineering and, homologous recombination. In the case of use with homologous recombination, no in vivo construct will be required.

The nucleic acid molecule may be a heterologous nucleic acid molecule. A heterologous polynucleotide or a heterologous nucleic acid or an exogenous DNA segment refers to a polynucleotide, nucleic acid or DNA segment that originates from a source foreign to the particular host cell, or, if from the same source, is modified from its original form in composition and/or genomic locus by human intervention. A heterologous gene in a host cell includes a gene that is endogenous to the particular host cell, but has been modified or introduced into the plant. Thus, the terms refer to a DNA segment which is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides.

In an example of a nucleic acid molecule or gene of interest, it may encode a protein that is useful for industrial or pharmaceutical purposes or the like, or to impact the plant itself, such as through expression of a protein that provides disease resistance, insect resistance, herbicide resistance, or impacts agronomic traits as well as grain quality traits. The sequences used with the promoter can be native or non-native sequences to the plant. DNA sequences native to plants as well as non-native DNA sequences can be transformed into plants and used to modulate levels of native or non-native proteins.

Plants have been used to produce a wide range of recombinant proteins of potential economic and/or medicinal importance. These include research chemicals (Hood et al., 1997; Zhong et al., 1999), processing enzymes that are used, for example, in the pharmaceutical industry (Woodard et al., 2003), industrial enzymes that are deployed in large-scale processing operations such as bleaching (Hood et al., 2003; Bailey et al., 2004), candidate vaccine antigens for animal or plant disease prevention (Mason et al., 1992; Haq et al., 1995; Carrillo et al., 1998; Streatfield et al., 2001), and therapeutic pharmaceuticals including antibodies (Daniell et al., 2001; Hood et al., 2002). The expressed proteins may either be purified from the plant tissues (Hood et al., 1997; Woodard et al., 2003) or, if as with vaccines the final application allows it, the recombinant plant material may be processed into a suitable form for use or even deployed directly (Streatfield et al., 2002; Lamphear et al., 2002). By way of example without limitation, in an embodiment the nucleic acid sequence may encode the hepatitis B surface antigen or aprotinin.

In another example the nucleic acid molecule encodes a cellulase. Expression of cellulases in plants can take many forms and any methods are useful with the multiple PTUs described. A description of various methods and cellulases is provided in US publication US20110143398, incorporated herein by reference in its entirety. Such enzymes may be used with cellulosic biomass in production of energy. By “cellulosic biomass” or feedstock is intended biomass that is comprised of plant cell walls and the components therein including, but not limited to, cellulose, hemicellulose, pectin, and lignin. Such cellulosic biomass includes, for example, crop plant residues or undesired plant material that may be left behind in the field after harvest or separated from the desired plant material or forest products or the like. A crop refers to a collection of plants grown in a particular cycle. By “desired plant material” is intended the plant product that is the primary reason for commercially growing the plant. Such desired plant material can be any plant or plant part or plant product that has commercial value. Corn is grown for human and animal consumption, as well as to produce products such as industrial oils, fertilizer and many other uses. Soybeans and wheat are used primarily in food products. There are multitudes of purposes for which these plant materials can be utilized. The desired plant material also includes protein produced by a transgenic polynucleotide. In short, the desired plant material refers to any product from the plant that is useful.

The multiple PTUs when used to encode a cellulase may be used in a process where breakdown of cellulose is desired. An example of such a process is described at US publication US20110143398. It allows for profitable use of desired plant material or what would otherwise be low value or waste material after the desired plant is harvested. What is more, one skilled in the art understands that the production of the plant material for use as plant tissue composition may in itself be production of desired plant material such as when crops such as switch grass are grown for the purpose of producing an energy source. In an embodiment of the invention, an enzyme substitute and/or an enzyme expressed as a heterologous protein in the plant can be used to degrade polysaccharides in a crop and can be produced by the very crop that will be degraded, thereby providing clear advantages in eliminating or reducing the need for an outside source of the enzyme, compacting costs with its production by combining it with production of the cellulose source. In addition, one skilled in the art can appreciate that the transgenic enzymes expressed in such plants may be used in any commercial polysaccharide-degrading process, such as in providing additives to animal feed (See, for example Rode et al., “Fibrolytic enzyme supplements for dairy cows in early lactation” J. Dairy Sci. 1999 October; 82(1):2121-6); industrial applications, (for example, in detergent applications, see Winetzky, U.S. Pat. No. 6,565,6131; in biofinishing of denims, see Vollmond, WO 97/25468); treatment of genes, or, in a preferred embodiment, in the production of ethanol.

The cellulases which may be produced in an embodiment may be useful for the conversion of plant cell wall polysaccharides to fermentable sugars that can then be used in the production of ethanol or other desired molecules via fermentation methods known in the art. The use of the term “fermentable sugars” includes, but is not limited to, monosaccharides and disaccharides and also encompasses sugar derivatives such as, for example, sugar alcohols, sugar acids, amino sugars, and the like. The fermentable sugars of the invention encompass any sugar or sugar derivative that is capable of being fermented using microorganisms. An example of production of endocellulases and exocellulases in plants is described at US Patent Publication No. 20060026715, incorporated herein by reference in its entirety. In an embodiment, the plant cell-produced heterologous protein can serve as the source of exogenous cellulose degrading enzyme. It can be purified if desired, or the plant cells or tissue comprising the heterologous enzyme added to the mixture. In one embodiment, the plant tissue composition transformed with one or more cellulose degrading enzymes can be the source of enhancement of the production of fermentable sugars by providing plant tissue composition for such enhancement, and also provide one or more exogenous cellulose degrading enzymes.

The enzymes used in saccharification processes and which may be used with the methods here described currently encompass enzymes that can be employed to degrade plant cell wall polysaccharides into fermentable sugars. Such enzymes are known in the art and include, but are not limited to, enzymes that can catalyze the degradation of cellulose, hemicellulose, and/or pectin. In particular, the methods of the invention are drawn to cellulose-degrading enzymes. By “cellulase” or “cellulose-degrading enzyme” is intended any enzyme that can be utilized to promote the degradation of cellulose into fermentable sugars including, but not limited to, cellulases and glucosidases. By way of example, without limitation, the enzymes classified in Enzyme Classification as 3.2.1.x are included within the scope of the invention. An example of the many enzymes which may be employed in the invention is presented in Table 1, a list of enzymes in the category by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB).

TABLE 1 Polysaccharide degrading enzymes   EC 3.2.1.1 α-amylase EC 3.2.1.2 β-amylase EC 3.2.1.3 glucan 1,4-α-glucosidase EC 3.2.1.4 cellulase EC 3.2.1.6 endo-1,3(4)-β-glucanase EC 3.2.1.7 inulinase EC 3.2.1.8 endo-1,4-β-xylanase EC 3.2.1.10 oligo-1,6-glucosidase EC 3.2.1.11 dextranase EC 3.2.1.14 chitinase EC 3.2.1.15 polygalacturonase EC 3.2.1.17 lysozyme EC 3.2.1.18 exo-α-sialidase EC 3.2.1.20 α-glucosidase EC 3.2.1.21 β-glucosidase EC 3.2.1.22 α-galactosidase EC 3.2.1.23 β-galactosidase EC 3.2.1.24 α-mannosidase EC 3.2.1.25 β-mannosidase EC 3.2.1.26 β-fructofuranosidase EC 3.2.1.28 αα-trehalase EC 3.2.1.31 β-glucuronidase EC 3.2.1.32 xylan endo-1,3-β-xylosidase EC 3.2.1.33 amylo-1,6-glucosidase EC 3.2.1.35 hyaluronoglucosaminidase EC 3.2.1.36 hyaluronoglucuronidase EC 3.2.1.37 xylan 1,4-β-xylosidase EC 3.2.1.38 β-D-fucosidase EC 3.2.1.39 glucan endo-1,3-β-D-glucosidase EC 3.2.1.40 β-L-rhamnosidase EC 3.2.1.41 pullulanase EC 3.2.1.42 GDP-glucosidase EC 3.2.1.43 β-L-rhamnosidase EC 3.2.1.44 fucoidanase EC 3.2.1.45 glucosylceramidase EC 3.2.1.46 galactosylceramidase EC 3.2.1.47 galactosylgalactosylglucosylceramidase EC 3.2.1.48 sucrose β-glucosidase EC 3.2.1.49 α-N-acetylgalactosaminidase EC 3.2.1.50 α-N-acetylglucosaminidase EC 3.2.1.51 α-L-fucosidase EC 3.2.1.52 β-L-N-acetylhexosaminidase EC 3.2.1.53 β-N-acetylgalactosaminidase EC 3.2.1.54 cyclomaltodextrinase EC 3.2.1.55 α-N-arabinofuranosidase EC 3.2.1.56 glucuronosyl-disulfoglucosamine glucuronidase EC 3.2.1.57 isopullulanase EC 3.2.1.58 glucan 1,3-β-glucosidase EC 3.2.1.59 glucan endo-1,3-α-glucosidase EC 3.2.1.60 glucan 1,4-α-maltotetraohydrolase EC 3.2.1.61 mycodextranase EC 3.2.1.62 glycosylceramidase EC 3.2.1.63 1,2-α-L-fucosidase EC 3.2.1.64 2,6-β-fructan 6-levanbiohydrolase EC 3.2.1.65 levanase EC 3.2.1.66 quercitrinase EC 3.2.1.67 galacturan 1,4-α-galacturonidase EC 3.2.1.68 isoamylase EC 3.2.1.70 glucan 1,6-α-glucosidase EC 3.2.1.71 glucan endo-1,2-β-glucosidase EC 3.2.1.72 xylan 1,3-β-xylosidase EC 3.2.1.73 licheninase EC 3.2.1.74 glucan 1,4-β-glucosidase EC 3.2.1.75 glucan endo-1,6-β-glucosidase EC 3.2.1.76 L-iduronidase EC 3.2.1.77 mannan 1,2-(1,3)-α-mannosidase EC 3.2.1.78 mannan endo-1,4-β-mannosidase EC 3.2.1.80 fructan β-fructosidase EC 3.2.1.81 agarase EC 3.2.1.82 exo-poly-α-galacturonosidase EC 3.2.1.83 κ-carrageenase EC 3.2.1.84 glucan 1,3-β-glucosidase EC 3.2.1.85 6-phospho-β-galactosidase EC 3.2.1.86 6-phospho-β-glucosidase EC 3.2.1.87 capsular-polysaccharide endo-1,3-α-galactosidase EC 3.2.1.88 β-L-arabinosidase EC 3.2.1.89 arabinogalactan endo-1,4-β-galactosidase EC 3.2.1.91 cellulose 1,4-β-cellobiosidase EC 3.2.1.92 peptidoglycan β-N-acetylmuramidase EC 3.2.1.93 αα-phosphotrehalase EC 3.2.1.94 glucan 1,6-α-isomaltosidase EC 3.2.1.95 dextran 1,6-α-isomaltotriosidase EC 3.2.1.96 mannosyl-glycoprotein endo-β-N-acetylglucosaminidase EC 3.2.1.97 glycopeptide α-N-acetylgalactosaminidase EC 3.2.1.98 glucan 1,4-α-maltohexaosidase EC 3.2.1.99 arabinan endo-1,5-α-L-arabinosidase EC 3.2.1.100 mannan 1,4-mannobiosidase EC 3.2.1.101 mannan endo-1,6-α-mannosidase EC 3.2.1.102 blood-group-substance endo-1,4-β-galactosidase EC 3.2.1.103 keratan-sulfate endo-1,4-β-galactosidase EC 3.2.1.104 sterl-β-glucosidase EC 3.2.1.105 strictosidine β-glucosidase EC 3.2.1.106 mannosyl-oligosaccharide glucosidase EC 3.2.1.107 protein-glucosylgalactosylhydroxylysine glucosidase EC 3.2.1.108 lactase EC 3.2.1.109 endogalactosaminidase EC 3.2.1.110 mucinaminylserine mucinaminidase EC 3.2.1.111 1,3-α-L-fucosidase EC 3.2.1.112 2-deoxyglucosidase EC 3.2.1.113 mannosyl-oligosaccharide 1,2-α-mannosidase EC 3.2.1.114 mannosyl-oligosaccharide 1,3-1,6-α-mannosidase EC 3.2.1.115 branched-dextran exo-1,2-α-glucosidase EC 3.2.1.116 glucan 1,4-α-maltotriohydrolase EC 3.2.1.117 amygdalin β-glucosidase EC 3.2.1.118 prunasin β-glucosidase EC 3.2.1.119 vicianin β-glucosidase EC 3.2.1.120 oligoxyloglucan β-glycosidase EC 3.2.1.121 polymannuronate hydrolase EC 3.2.1.122 maltose-6′-phosphate glucosidase EC 3.2.1.123 endoglycosylceramidase EC 3.2.1.124 3-deoxy-2-octulosonidase EC 3.2.1.125 raucaffricine β-glucosidase EC 3.2.1.126 coniferin β-glucosidase EC 3.2.1.127 1,6-α-L-fucosidase EC 3.2.1.128 glycyrrhizinate β-glucuronidase EC 3.2.1.129 endo-α-sialidase EC 3.2.1.130 glycoprotein endo-α-1,2-mannosidase EC 3.2.1.131 xylan α-1,2-glucuronosidase EC 3.2.1.132 chitosanase EC 3.2.1.133 glucan 1,4-α-maltohydrolase EC 3.2.1.134 difructose-anhydride synthase EC 3.2.1.135 neopullulanase EC 3.2.1.136 glucuronoarabinoxylan endo-1,4-β-xylanase EC 3.2.1.137 mannan exo-1,2-1,6-β-mannosidase EC 3.2.1.139 α-glucuronidase EC 3.2.1.140 lacto-N-biosidase EC 3.2.1.141 4-α-D-((1 → 4)-α-D-glucano)trehalose trehalohydrolase EC 3.2.1.142 limit dextrinase EC 3.2.1.143 poly(ADP-ribose) glycohydrolase EC 3.2.1.144 3-deoxyoctulosonase EC 3.2.1.145 galactan 1,3-β-galactosidase EC 3.2.1.146 β-galactofuranosidase EC 3.2.1.147 thioglucosidase EC 3.2.1.149 β-primeverosidase EC 3.2.1.150 oligoxyloglucan reducing-end-specific cellobiohydrolase EC 3.2.1.151 xyloglucan-specific endo-β-1,4-glucanase EC 3.2.1.152 mannosylglycoprotein endo-β-mannosidase EC 3.2.1.153 fructan β-(2,1)-fructosidase EC 3.2.1.154 fructan β-(2,6)-fructosidase EC 3.2.1.156 oligosaccharide reducing-end xylanase

For the degradation of cellulose, two types of exoglucanase have been described that differ in their approach to the cellulose chain. One type attacks the non-reducing end and the other attacks the reducing end. Cellulase enzymes which cleave the cellulose chain internally are referred to as endo-β-1,4-glucanases (E.C. 3.2.1.4) and serve to provide new reducing and non-reducing chain termini on which exo-β-1,4-glucanases (cellobiohydrolase, CBH; E.C. 3.2.1.91) can operate (Tomme et al. (1995) Microbial Physiology 37:1-81). The product of the exoglucanase reaction is typically cellobiose, so a third activity, β-D-glucosidase (E.C. 3.2.1.21), is required to cleave cellobiose to glucose. The exoglucanase can also yield longer glucose chains (up to 6 glucose units) that will require a β-D-glucosidase activity to reduce their size. Relative to the other enzyme activities needed for degradation of cellulose into fermentable sugars, only a minor amount of the β-D-glucosidase activity is required. In brief, current processes to produce fermentable sugars involve the addition to a cellulose-containing composition an endocellulase (endo-β-1,4-glucanases) and an exocellulase (exo-β-1,4-glucanases) which cleaves the cellulose chain internally. In order to produce the end product of glucose, a third enzyme is involved, a glucosidase (β-D-glucosidases), which acts on the cellobiose to produce glucose. One skilled understands that other proteins can increase the rate as, for example, expansins, which unfold the crystalline cellulose to make it more available so the enzymes can degrade it more efficiently. Cosgrove (1999) Annu Rev Plant Physiol Plant Mol Biol 50:391-417.

If desired, the gene of interest can be optimized for plant translation by optimizing the codons used for plants and the sequence around the translational start site for plants. Sequences resulting in potential mRNA instability can also be avoided.

Clearly, one skilled in the art appreciates there can be variations in the promoter or nucleic acid sequence tolerated and still produce the increased expression described. Identity to a sequence described can be a polynucleotide sequence having at least 65% sequence identity, more preferably at least 70% sequence identity, more preferably at least 75% sequence identity, more preferably at least 80% identity, and more preferably at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with a sequence exemplified or described herein. Hybridization and hybridization conditions as provided herein can also be used to define polynucleotide sequences of the invention.

The nucleotide sequences of the promoter or nucleic acid molecule can be used to isolate corresponding sequences from other organisms, particularly other plants, or to synthesize synthetic sequences. In this manner, methods such as PCR, hybridization, synthetic gene construction and the like can be used to identify or generate such sequences based on their sequence homology to the sequences set forth herein. Sequences identified, isolated or constructed based on their sequence identity to the whole of or any portion of the original sequence may be used in the present invention. In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any plant of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed (Sambrook et al., 1989; Innis et al., 1990; Innis et al., 1995; Innis et al., 1999). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers, and the like.

In hybridization techniques, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as ³²P, or any other detectable marker. Thus, for example, probes for hybridization can be made by labeling synthetic oligonucleotides based on the DNA sequences of the invention. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed (Sambrook et al., 1989).

For example, the sequence, or one or more portions thereof, may be used as a probe capable of specifically hybridizing to corresponding sequences. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique among the sequences to be screened and are preferably at least about 10 nucleotides in length, and most preferably at least about 20 nucleotides in length. Such sequences may alternatively be used to amplify corresponding sequences from a chosen plant by PCR. This technique may be used to isolate sequences from a desired plant or as a diagnostic assay to determine the presence of sequences in a plant. Hybridization techniques include hybridization screening of DNA libraries plated as either plaques or colonies (Sambrook et al., 1989).

Hybridization of such sequences may be carried out under stringent conditions. By “stringent conditions” or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C.

Specificity is also the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the T_(m) can be approximated from the equation T_(m)=81.5° C.+16.6 (logM)+0.41(% GC)−0.61(% form.)−500/L, where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form. is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs (Meinkoth and Wahl, 1984). The T_(m) is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C. for each 1% of mismatching; thus, T_(m), hybridization, and/or wash conditions can be adjusted for sequences of the desired identity to hybridize. For example, if sequences with 90% identity are sought, the T_(m) can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (T_(m)) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point (T_(m)); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (T_(m)); low stringency conditions can utilize a hybridization and/or wash at 11 to 20° C. lower than the thermal melting point (T_(m)). Using the equation, hybridization and wash compositions, and desired T_(m), those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T_(m) of less than 45° C. (aqueous solution) or 32° C. (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Ausubel et al. (1993) and Sambrook et al. (1989).

Thus, isolated sequences that have promoter activity and which hybridize under stringent conditions to the promoter sequences disclosed herein, or to fragments thereof, are encompassed by the present invention.

The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity” and (d) “percentage of sequence identity.”

(a) As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length promoter sequence, or the complete promoter sequence.

(b) As used herein, “comparison window” makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to accurately reflect the similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm. Optimal alignment of sequences for comparison can use any means to analyze sequence identity (homology) known in the art, e.g., by the progressive alignment method of termed “PILEUP” (Morrison, Mol. Biol. Evol. 14:428-441 (1997), as an example of the use of PILEUP); by the local homology algorithm of Smith & Waterman (Adv. Appl. Math. 2: 482 (1981)); by the homology alignment algorithm of Needleman & Wunsch (J. Mol. Biol. 48:443 (1970)); by the search for similarity method of Pearson (Proc. Natl. Acad. Sci. USA 85: 2444 (1988)); by computerized implementations of these algorithms (e.g., GAP, BEST FIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.); ClustalW (CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif., described by, e.g., Higgins, Gene 73: 237-244 (1988); Corpet, Nucleic Acids Res. 16:10881-10890 (1988); Huang, Computer Applications in the Biosciences 8:155-165 (1992); and Pearson, Methods in Mol. Biol. 24:307-331 (1994); Pfam (Sonnhammer, Nucleic Acids Res. 26:322-325 (1998); TreeAlign (Hein, Methods Mol. Biol. 25:349-364 (1994); MEG-ALIGN, and SAM sequence alignment computer programs; or, by manual visual inspection.

Another example of algorithm that is suitable for determining sequence similarity is the BLAST algorithm, which is described in Altschul et al, J. Mol. Biol. 215: 403-410 (1990). The BLAST programs (Basic Local Alignment Search Tool) of Altschul, S. F., et al., (1993) J. Mol. Biol. 215:403-410) searches under default parameters for identity to sequences contained in the BLAST “GENEMBL” database. A sequence can be analyzed for identity to all publicly available DNA sequences contained in the GENEMBL database using the BLASTN algorithm under the default parameters.

Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information, www.ncbi.nlm.nih.gov/; see also Zhang, Genome Res. 7:649-656 (1997) for the “PowerBLAST” variation. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, J. Mol. Biol. 215: 403-410 (1990)). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919 (1992)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands. The term BLAST refers to the BLAST algorithm that performs a statistical analysis of the similarity between two sequences; see, e.g., Karlin, Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

In an embodiment, GAP (Global Alignment Program) can be used. GAP uses the algorithm of Needleman and Wunsch J. Mol. Biol. 48:443-453 (1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. Default gap creation penalty values and gap extension penalty values in the commonly used Version 10 of the Wisconsin Package® (Accelrys, Inc., San Diego, Calif.) for protein sequences are 8 and 2, respectively. For nucleotide sequences the default gap creation penalty is 50 while the default gap extension penalty is 3. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. A general purpose scoring system is the BLOSUM62 matrix (Henikoff and Henikoff, Proteins, 17: 49-61 (1993)), which is currently the default choice for BLAST programs. BLOSUM62 uses a combination of three matrices to cover all contingencies. Altschul, J. Mol. Biol. 36: 290-300 (1993), herein incorporated by reference in its entirety and is the scoring matrix used in Version 10 of the Wisconsin Package® (Accelrys, Inc., San Diego, Calif.) (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

As used herein, “sequence identity” or “identity” in the context of two nucleic acid sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.

As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

Identity to the sequence of the present invention would mean a polynucleotide sequence having at least 65% sequence identity, more preferably at least 70% sequence identity, more preferably at least 75% sequence identity, more preferably at least 80% identity, more preferably at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity.

“Functional variants” of the sequence disclosed may be used. Functional variants include, for example, sequences having one or more nucleotide substitutions, deletions or insertions and wherein the variant retains promoter activity, particularly the ability to drive expression preferentially to the embryo of a plant, or encodes the product. Functional variants can be created by any of a number of methods available to one skilled in the art, such as by site-directed mutagenesis, induced mutation, identified as allelic variants, cleaving through use of restriction enzymes, or the like. Activity can likewise be measured by any variety of techniques, including measurement of reporter activity as is described at U.S. Pat. No. 6,844,484, Northern blot analysis or similar techniques.

Further, a “functional fragment” may be used, that is a regulatory fragment formed by one or more deletions from a larger regulatory element, or a fragment of a nucleic acid sequence that encodes a desired product. For example, the 5′ portion of a promoter up to the TATA box near the transcription start site can be deleted without abolishing promoter activity, as described by Opsahl-Sorteberg, H-G. et al., 2004. Such fragments should retain promoter activity, particularly the ability to drive expression of operably linked nucleotide sequences. Activity can be measured by Northern blot analysis, reporter activity measurements when using transcriptional fusions, and the like. See for example, Sambrook et al. (1989). Functional fragments can be obtained by use of restriction enzymes to cleave the naturally occurring regulatory element nucleotide sequences disclosed herein; by synthesizing a nucleotide sequence from the naturally occurring DNA sequence; or can be obtained through the use of PCR technology. See particularly, Mullis et al. (1987) and Erlich, ed. (1989).

For example, a routine way to remove a part of a DNA sequence is to use an exonuclease in combination with DNA amplification to produce unidirectional nested deletions of double stranded DNA clones. A commercial kit for this purpose is sold under the trade name Exo-Size™ (New England Biolabs, Beverly, Mass.). Briefly, this procedure entails incubating exonuclease III with DNA to progressively remove nucleotides in the 3′ to 5′ direction at the 5′ overhangs, blunt ends or nicks in the DNA template. However, the exonuclease III is unable to remove nucleotides at 3′ 4-base overhangs. Timed digest of a clone with this enzyme produces unidirectional nested deletions.

Once the gene is engineered to contain desired features, such as the desired subcellular localization sequences, it may then be placed into an expression vector by standard methods. The selection of an appropriate expression vector will depend upon the method of introducing the expression vector into host cells. A typical expression vector contains prokaryotic DNA elements coding for a bacterial origin of replication and an antibiotic resistance gene to provide for the growth and selection of the expression vector in the bacterial host; a cloning site for insertion of an exogenous DNA sequence; eukaryotic DNA elements that control initiation of transcription of the exogenous gene (such as the promoter of the invention or another promoter); and DNA elements that control the processing of transcripts, such as transcription termination/polyadenylation sequences. It also can contain such sequences as are needed for the eventual integration of the vector into the plant chromosome.

One skilled in the art will appreciate that various other components may be included in a vector used with the multiple PTUs and will vary depending on the specific application. In general, the methods available for construction of recombinant genes, optionally comprising various modifications for improved expression, can differ in detail. However, conventionally employed methods include PCR amplification, or the designing and synthesis of overlapping, complementary synthetic oligonucleotides, which are annealed and ligated together to yield a gene with convenient restriction sites for cloning, or subcloning from another already cloned source, or cloning from a library. The methods involved are standard methods for a molecular biologist (Sambrook et al., 1989).

In one embodiment, the expression vector may also contain a gene encoding a selectable or scoreable marker that is operably or functionally linked to a promoter that controls transcription initiation, which can be the promoter of the invention or another promoter. By “operably linked” it is understood that the gene of interest is oriented in connection to the gene such that the promoter initiates transcription of the gene in order to allow its expression of the resulting protein in plants. For a general description of plant expression vectors and reporter genes, see Gruber et al. (1993). For example, the selective gene may be a glufosinate-resistance encoding DNA and in another example it can be phosphinothricin acetyl transferase (pat) or a maize optimized pat gene under the control of the CaMV 35S promoter. Such pat genes confer resistance to the herbicide bialaphos (Gordon-Kamm et al., 1990).

The multiple PTUs driving the gene of interest may be used in conjunction with other promoters used to drive other nucleotide sequences than the gene of interest where desired. In one embodiment, a plant selection marker and the gene of interest can be both functionally linked to the same promoter. In another embodiment, a plant selection marker and the gene of interest can be functionally linked to different promoters. These other promoter elements can be those that are constitutive or sufficient to render promoter-dependent gene expression controllable as being cell-type specific, tissue-specific or time or developmental stage specific, or being inducible by external signals or agents. Such elements may be located in the 5′ or 3′ regions of the gene. Although the additional promoter may be the endogenous promoter of a structural gene of interest, the promoter can also be a foreign regulatory sequence. Promoter elements employed to control expression of product proteins and the selection gene can be any plant-compatible promoters.

The expression vector can optionally also contain a signal sequence located between the promoter and the gene of interest. A signal sequence is a nucleotide sequence, translated to give an amino acid sequence, which is used by a cell to direct the protein or polypeptide of interest to be placed in a particular place within or outside the eukaryotic cell. One example of a plant signal sequence is the barley α-amylase secretion signal (Rogers, 1985). Many signal sequences are known in the art. See, for example Becker et al. (1992), Fontes et al. (1991), Matsuoka and Nakamura (1991), Gould et al. (1989), Creissen et al. (1992), Kalderon et al. (1984) and Stiefel et al. (1990).

Leader sequences can be included to enhance translation. Instead of, or in addition to the untranslated leader sequence of the promoter, other leader sequences may be substituted or added. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995)); human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991)); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987)); tobacco mosaic virus leader (TMV) (Gallie. (1989)); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991)). See also, Della-Cioppa et al. (1987). Other methods known to enhance translation can also be utilized, for example, introns, and the like. Obviously, many variations on the promoters, selectable markers, signal sequences, leader sequences, termination sequences, introns, enhancers and other components of the construct are available to one skilled in the art.

It is anticipated the invention can be used with monocotyledonous or dicotyledonous plants. Examples of monocotyledonous plants are plants which belong to the genus of avena (oat), triticum (wheat), secale (rye), hordeum (barley), oryza (rice), panicum, pennisetum, setaria, sorghum (millet), zea (maize). Dicotyledonous useful plants are, inter alia, leguminous plants, such as legumes and especially alfalfa, soybean, rape, tomato, sugar beet, and potato.

The PTUs of the invention may be introduced into any plant or plant part. The term plant or plant material or plant part is used broadly herein to include any plant at any stage of development, or to part of a plant, including a plant cutting, a plant cell, a plant cell culture, a plant organ, a plant seed, and a plantlet. Examples of such plant parts are plant cells, embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, silk, flowers, kernels, ears, cobs, husks or stalks. A plant cell is the structural and physiological unit of the plant, comprising a protoplast and a cell wall. A plant cell can be in the form of an isolated single cell or aggregate of cells such as a friable callus, or a cultured cell, or can be part of a higher organized unit, for example, a plant tissue, plant organ, or plant. Thus, a plant cell can be a protoplast, a gamete producing cell, or a cell or collection of cells that can regenerate into a whole plant. As such, a seed, which comprises multiple plant cells and is capable of regenerating into a whole plant, is considered a plant cell for purposes of this disclosure. A plant tissue or plant organ can be a seed, protoplast, callus, or any other groups of plant cells that is organized into a structural or functional unit. Particularly useful parts of a plant include harvestable parts and parts useful for propagation of progeny plants. A harvestable part of a plant can be any useful part of a plant, for example, flowers, pollen, seedlings, tubers, leaves, stems, fruit, seeds, roots, and the like. A part of a plant useful for propagation includes, for example, seeds, fruits, cuttings, seedlings, tubers, rootstocks, and the like. Still further, the present invention provides plants regenerated from the tissue cultures of the invention.

Methods for introducing expression vectors into plant tissue available to one skilled in the art are varied and will depend on the plant selected. Procedures for transforming a wide variety of plant species are well known and described throughout the literature. See, for example, Mild and McHugh (2004); Klein et al. (1992); and Weising et al. (1988). For example, the DNA construct may be introduced into the genomic DNA of the plant cell using techniques such as microprojectile-mediated delivery (Klein et al. 1992), electroporation (Fromm et al., 1985), polyethylene glycol (PEG) precipitation (Mathur and Koncz, 1998), direct gene transfer (WO 85/01856 and EP-A-275 069), in vitro protoplast transformation (U.S. Pat. No. 4,684,611) and microinjection of plant cell protoplasts or embryogenic callus (Crossway, 1985). Co-cultivation of plant tissue with Agrobacterium tumefaciens is another option, where the DNA constructs are placed into a binary vector system (Ishida et al., 1996). The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct into the plant cell DNA when the cell is infected by the bacteria. See, for example, Fraley et al. (1983).

Standard methods for transformation of canola are described by Moloney et al. (1989). Corn transformation is described by Fromm et al. (1990) and Gordon-Kamm et al. (1990). Agrobacterium is primarily used in dicots, but certain monocots such as maize can be transformed by Agrobacterium. See, for example, U.S. Pat. No. 5,550,318. Rice transformation is described by Hiei et al. (1994) and Lee et al. (1991). Wheat can be transformed by techniques similar to those used for transforming corn or rice. Sorghum transformation is described by Casas et al. (1993) and barley transformation is described by Wan and Lemaux (1994). Soybean transformation is described in a number of publications, including U.S. Pat. No. 5,015,580.

In one method, the Agrobacterium transformation methods of Ishida et al. (1996) and also described in U.S. Pat. No. 5,591,616, are generally followed, with modifications that the inventors have found improve the number of transformants obtained. The Ishida method uses the A188 variety of maize that produces Type I callus in culture. In one preferred embodiment the Hi II maize line is used which initiates Type II embryogenic callus in culture (Armstrong et al., 1991).

While Ishida recommends selection on phosphinothricin when using the bar or pat gene for selection, another preferred embodiment provides use of bialaphos instead. In general, as set forth in the 5,591,616 patent, and as outlined in more detail below, dedifferentiation is obtained by culturing an explant of the plant on a dedifferentiation-inducing medium for not less than seven days, and the tissue during or after dedifferentiation is contacted with Agrobacterium having the gene of interest. The cultured tissue can be callus, an adventitious embryo-like tissue or suspension cells, for example. In this preferred embodiment, the suspension of Agrobacterium has a cell population of 10⁶ to 10¹¹ cells/ml and are contacted for three to ten minutes with the tissue, or continuously cultured with Agrobacterium for not less than seven days. The Agrobacterium can contain plasmid pTOK162, with the gene of interest between border sequences of the T region of the plasmid, or the gene of interest may be present in another plasmid-containing Agrobacterium. The virulence region may originate from the virulence region of a Ti plasmid or Ri plasmid. The bacterial strain used in the Ishida protocol is LBA4404 with the 40 kb super binary plasmid containing three vir loci from the hypervirulent A281 strain. The plasmid has resistance to tetracycline. The cloning vector cointegrates with the super binary plasmid. Since the cloning vector has an E. coli specific replication origin, but not an Agrobacterium replication origin, it cannot survive in Agrobacterium without cointegrating with the super binary plasmid. Since the LBA4404 strain is not highly virulent, and has limited application without the super binary plasmid, the inventors have found in yet another embodiment that the EHA101 strain is preferred. It is a disarmed helper strain derived from the hypervirulent A281 strain. The cointegrated super binary/cloning vector from the LBA4404 parent is isolated and electroporated into EHA101, selecting for spectinomycin resistance. The plasmid is isolated to assure that the EHA101 contains the plasmid. EHA101 contains a disarmed pTi that carries resistance to kanamycin. See, Hood et al. (1986).

Further, the Ishida protocol as described provides for growing fresh culture of the Agrobacterium on plates, scraping the bacteria from the plates, and resuspending in the co-culture medium as stated in the 5,591,616 patent for incubation with the maize embryos. This medium includes 4.3 g MS salts, 0.5 mg nicotinic acid, 0.5 mg pyridoxine hydrochloride, 1.0 ml thiamine hydrochloride, casamino acids, 1.5 mg 2,4-D, 68.5 g sucrose and 36 g glucose per liter, all at a pH of 5.8. In a further preferred method, the bacteria are grown overnight in a 1 ml culture and then a fresh 10 ml culture is re-inoculated the next day when transformation is to occur. The bacteria grow into log phase, and are harvested at a density of no more than OD₆₀₀=0.5, preferably between 0.2 and 0.5. The bacteria are then centrifuged to remove the media and resuspended in the co-culture medium. Since Hi II is used, medium preferred for Hi II is used. This medium is described in considerable detail by Armstrong and Green (1985). The resuspension medium is the same as that described above. All further Hi II media are as described in Armstrong and Green (1985). The result is redifferentiation of the plant cells and regeneration into a plant. Redifferentiation is sometimes referred to as dedifferentiation, but the former term more accurately describes the process where the cell begins with a form and identity, is placed on a medium in which it loses that identity, and becomes “reprogrammed” to have a new identity. Thus the scutellum cells become embryogenic callus.

In accordance with the present invention, a transgenic plant is produced that contains an introduced multiple PTU. It can be combined with any one of the components set forth above.

In a further embodiment, plant breeding can be used to introduce the nucleotide sequences into other plants once transformation has occurred. This can be accomplished by any means known in the art for breeding plants such as, for example, cross pollination of the transgenic plants that are described above with other plants, and selection for plants from subsequent generations which express the amino acid sequence. The plant breeding methods used herein are well known to one skilled in the art. For a discussion of plant breeding techniques, see Poehlman and Sleper (1995). Many crop plants useful in this method are bred through techniques that take advantage of the plant's method of pollination. A plant is self-pollinating if pollen from one flower is transferred to the same or another flower of the same plant. A plant is cross-pollinating if the pollen comes from a flower on a different plant. For example, in Brassica, the plant is normally self-sterile and can only be cross-pollinated unless, through discovery of a mutant or through genetic intervention, self-compatibility is obtained. In self-pollinating species, such as rice, oats, wheat, barley, peas, beans, soybeans, tobacco and cotton, the male and female plants are anatomically juxtaposed. During natural pollination, the male reproductive organs of a given flower pollinate the female reproductive organs of the same flower. Maize plants (Zea mays L.) can be bred by both self-pollination and cross-pollination techniques. Maize has male flowers, located on the tassel, and female flowers, located on the ear, on the same plant. It can self or cross-pollinate.

Pollination can be by any means, including but not limited to hand, wind or insect pollination, or mechanical contact between the male fertile and male sterile plant. For production of hybrid seeds on a commercial scale in most plant species pollination by wind or by insects is preferred. Stricter control of the pollination process can be achieved by using a variety of methods to make one plant pool male sterile, and the other the male fertile pollen donor. This can be accomplished by hand detas sling, cytoplasmic male sterility, or control of male sterility through a variety of methods well known to the skilled breeder. Examples of more sophisticated male sterility systems include those described by Brar et al., U.S. Pat. Nos. 4,654,465 and 4,727,219 and Albertsen et al., U.S. Pat. Nos. 5,859,341 and 6,013,859.

Backcrossing methods may be used to introduce the gene into the plants. This technique has been used for decades to introduce traits into a plant. An example of a description of this and other plant breeding methodologies that are well known can be found in references such as Neal (1988). In a typical backcross protocol, the original variety of interest (recurrent parent) is crossed to a second variety (nonrecurrent parent) that carries the single gene of interest to be transferred. The resulting progeny from this cross are then crossed again to the recurrent parent and the process is repeated until a plant is obtained wherein essentially all of the desired morphological and physiological characteristics of the recurrent parent are recovered in the converted plant, in addition to the single transferred gene from the nonrecurrent parent.

A variety of assays for the presence of and determine the expression level of the product encoded by the nucleic acid molecule are well known to a person skilled in the art. Methods to detect presence of E1 and CBH1 in extracts prepared from callus and seeds of plants having the heterologous protein, for example, are known. See, Coughlan et al. ((1988) J. Biol. Chem. 263:16631-16636) and Freer ((1993) J. Biol. Chem. 268:9337-9342). In addition, Western analysis and ELISAs can be used to assess protein integrity and expression levels. Individual T₁ seeds are screened by the assay of choice for expression of the target protein. Plants having homozygous condition of the transgenic construct expressing the protein, that is more than one copy of the gene, are expected to have increased expression levels of the enzyme. Expression levels of two to three to four fold or more are expected. It is expected that certain germplasm may have higher levels of expression of the enzyme and may also be selected. The individual plants expressing the highest levels of active enzyme are chosen for field studies, which include back-crosses (See “Plant Breeding Methodology” edit. Neal Jensen, John Wile & Sons, Inc. 1988), selection for increased expression and increased seed amounts. As is evident to one skilled in the art, it is possible to use the processes described to produce a biomass of transformed plants, select higher or highest expressing plant(s), and from selected plant(s) may produce a further biomass of plants expressing the desired protein at higher levels and thus provide a convenient source of the protein.

A Western analysis is a variation of the Southern analysis technique. With a Southern analysis, DNA is cut with restriction endonucleases and fractionated on an agarose gel to separate the DNA by molecular weight and then transferring to nylon membranes. It is then hybridized with the probe fragment which was radioactively labeled with ³²P and washed in an SDS solution. In the Western analysis, instead of isolating DNA, the protein of interest is extracted and placed on an acrylamide gel. The protein is then blotted onto a membrane and contacted with a labeling substance. See e.g., Hood et al., “Commercial Production of Avidin from Transgenic Maize; Characterization of Transformants, Production, Processing, Extraction and Purification” Molecular Breeding 3:291-306 (1997).

The ELISA or enzyme linked immunoassay has been known since 1971. In general, antigens solubilised in a buffer are coated on a plastic surface. When serum is added, antibodies can attach to the antigen on the solid phase. The presence or absence of these antibodies can be demonstrated when conjugated to an enzyme. Adding the appropriate substrate will detect the amount of bound conjugate which can be quantified. A common ELISA assay is one which uses biotinylated anti-(protein) polyclonal antibodies and an alkaline phosphatase conjugate. For example, an ELISA used for quantitative determination of laccase levels can be an antibody sandwich assay, which utilizes polyclonal rabbit antibodies obtained commercially. The antibody is conjugated to alkaline phosphatases for detection. In another example, an ELISA assay to detect trypsin or trypsinogen uses biotinylated anti-trypsin or anti-trypsinogen polyclonal antibodies and a streptavidin-alkaline phosphatase conjugate.

An initial test of enzymatic function in one embodiment is performed with lines of processed corn seed. For saccharification of cellulose, plant tissue from these lines are mixed in the appropriate ratio to produce a high specific activity for degradation of crystalline cellulose. According to Baker et al. ((1995) “Synergism between purified bacterial and fungal cellulases”, in Enzymatic Degradation of Insoluble Carbohydrates. ACS Series 618, American Chemical Society, Washington, D.C., pp. 113-141.), in an embodiment, maximum synergism for saccharification of cellulose is with a composite that is about 80% of the Trichoderma reesei CBHI (exo-β-1,4-glucanase) and about 20% of the Acidothermus cellulolyticus endo-β-1,4-glucanase. The addition of about 0.1% of the Candida wickerhamii β-D-glucosidase facilitates the degradation of short glucose oligomers (dp=2-6) to yield glucose. In transgenic enzyme production, later, cross pollination of the selected lines can be used to produce lines that express all three of the cellulase-degrading enzymes or these different enzymes can be engineered into one construct which in turn is transformed into the plant.

The product produced may be used in any convenient manner, whether the protein is extracted, and extract prepared as described below, or tissue or plant parts or plants comprising the product utilized. When referring to a plant tissue composition is meant any plant part, plant tissue (which can be optionally ground, sieved, pulverized, chopped, sliced, minced, ground, crushed, mashed or soaked or the like as long as the desired property is retained) or extract. Further, it is not meant to imply the entire plant must be used or that plant tissue or cells must be present in the composition in the final extract where an extract is provided as long as plant cells are used to produce the extract. For example, plant seed, leaves, roots, stem or other plant parts and tissue of plant parts and extracts of same can be employed in an embodiment of the invention. Seed tissue can include the whole seed or its parts, including pericarp (kernel or hull), embryo (called the germ in processing language), or endosperm. In an embodiment, the plant tissue is embryo plant tissue or extract. The plant seed tissue may be in another embodiment a grain seed or part thereof. In yet another embodiment, the plant tissue is a corn seed tissue or part thereof, such as, for example, an embryo that is also referred to as the germ. When referring to tissue is meant an aggregate of cells that can constitute structure(s) or component(s) of the plant, or which can be a portion of such structure or component, or which are from more than one such structure or organ. Seed tissue can be whole seed, portions of the seed, and ground or pulverized or otherwise processed in a manner that is convenient. The tissue composition in one embodiment can be a suspension of plant cells. As has been noted, whole plant may be used where convenient for the process, though one may desire to instead use other plant parts for other profitable uses.

The tissue composition can also be provided as an extract. While it may be convenient to provide tissue in the form of plant parts, whole seed or seed components, for example, one may also prepare flour or the like, there may be instances in which use of an extract is desired. Any of the many available means to prepare such an extract can be employed. When referring to an extract is meant the general process of placing tissue/cells in a liquid, preferably a buffer (the tissue may be optionally ground or otherwise pre-treated), and removing the supernatant. In one embodiment, the tissue may be in the liquid, without the need to purify a single protein. The supernatant may be further passed through a desalting column to separate high molecular weight from low molecular weight compounds, and the high molecular weight fraction used. However, in a commercial situation, the presence of glucose could be highly desirable. Thus one can prepare an extract in those situations where it is convenient to do so, by simple placing tissue in a liquid. A person skilled in the art could test any such extract for use in the invention by determining if it provides the increase in fermentable sugars and/or synergist result found here. For example, one may test the extract by determining if it increases production of fermentable sugars from cellulose when added with a combination of endocellulase, exocellulase and β-D glucosidase (such as, for example, the commercially available Spezyme® composition) and determining if release of fermentable sugars is at least 25% higher compared to the same process where the extract is not added. Further, one could test to determine if fermentable sugars are released when the extract is combined with cellulose, with reduced amounts of cellulase, or with no additional β-D glucosidase added. In another embodiment, one could test to determine if glucose is produced when the extract is combined with cellulose and no additional cellulase or β-D glucosidase is added.

The following is presented by way of illustration and is not intended to be limiting.

EXAMPLES Example 1

To obtain high expression of cellulase in the germ, a promoter derived from the globulin-1 storage protein (Belanger, 1989, Kriz, 1989) has been used (Hood, 2007). Recently, several additional strong embryo-preferred promoters including those of the globulin-2 gene and two genes of unknown function (pr36 and pr26) were identified with the hope that these would be useful to confer high levels of recombinant protein expression in maize (Streatfield, 2010). The globulin-2 gene is that shown at GenBank accession number AR947679 (SEQ ID NO: 8). To assess the use of these promoters to increase cellulase expression in transgenic maize, a series of constructs expressing either E1 (from Acidothermus cellulolyticus) or CBHI (from Trichoderma reesei) under control of various combinations of these promoters were prepared. This combination of E1 and CBHI was chosen because they exhibit synergistic activity on lignocellulosic substrates, high temperature optima, and compatible pH optima (Mohagherghi 1986, Nieves 1995, Shoemaker 1983, Baker 1998, Hood 2007).

Results

As shown below, use of transcription units driven by repeats of the same promoter and also where driven by different multiple promoters can increase expression. This is useful, as demonstrated, with an endocellulase, that is an endo-β-1,4-glucanase (E1, E.C. 3.2.1.4), and with an exocellulase, that is an exo-β-1,4-glucanase (cellobiohydrolase, CBH; E.C. 3.2.1.91).

The 1.4 kb globulin-1 promoter is the globulin-1 promoter described by Belanger and Kriz (1991) (here, SEQ ID NO: 1) and in the experiments below this specific promoter used included 43 extra base pairs (SEQ ID NO: 4) which are not a part of the promoter. The 3 kb extended globulin-1 is the extended globulin-1 promoter (SEQ ID NO: 5), incorporated herein by reference in its entirety. The globulin-2 promoter is shown at SEQ ID NO: 8 and the specific sequence used in the experiments described below has a minor variation in that the G nucleotide at position 279 is deleted, and the A nucleotide at position 728 is replaced by a C (this variation is SEQ ID NO: 20). The pr26 promoter used is SEQ ID NO: 11 and the specific sequence used in the experiments below has a minor variation in that the T directly preceding the ATG translation start site was deleted and an extra CACC sequence was inserted at this location. The pr36 promoter used is SEQ ID NO: 14 and the specific sequence used in the experiment below has a minor variation in that there is an extra GGGCAACC directly preceding the ATG translation start site (this variation is SEQ ID NO: 21). The extra regions are inconsequential variations on the promoters.

Constructs were made with either the E1 or CBHI cellulase enzyme under the control of the embryo-preferred promoters as shown in FIGS. 6 a and 6 b. One series included multiple copies of a transcription unit driven by the 3 kb maize globulin-1 promoter. While adding additional copies of the same transcription unit may increase expression, a concern with this approach is that it may be prone to lowered expression due to recombination or gene silencing. Thus, constructs with three separate transcription units driven by three different promoters were also prepared for comparison. For E1 the globulin-1, globulin-2, and pr26 promoters were used. The pr26 promoter is found at GenBank accession No. EA076965. For CBHI the globulin-1, globulin-2, and pr36 promoters were used. In previous work it was found that E1 was expressed at the highest level when targeted to the vacuole but was also strongly expressed in the endoplasmic reticulum (Hood, 2007). Thus a construct with one copy of E1 targeted to the vacuole and one targeted to the ER was prepared. Each of the constructs was used to transform maize and multiple events were obtained based on resistance to bialaphos. Multiple plants from independent transformation events were regenerated and a summary of the seed obtained available for biochemical analysis is shown in Table 2.

TABLE 2 Number of transformation events and plants analyzed for each construct Independent transformation Total number Construct events of plants CEA  6 45 CEB 10 72 CEC  8 44 CEJ  8 51 CEK 11 60 CEL  9 70 BCU  9 61 CCA  9 48 CCF 13 56 CCG 14 67

Cellulase expression was assessed using a biochemical assay with 4-methyl umbelliferyl cellobioside (MUC) as the substrate. Because of the limited availability of purified enzymes, it was not possible to obtain absolute amounts of the enzymes in routine assays, therefore a relative value was calculated. For T₁ seed extracts the florescence units (FU) per μg total soluble protein (TSP) assayed was determined. To take into account any variation between assays a positive control using the microbial produced T. reesei was used to normalized the fluorescence units run on different days.

The 3 kb globulin-1 promoter was previously found to support a two-fold greater expression of the GUS reporter gene compared to the 1.4 kb globulin1 promoter (Streatfield 2010). In this study, the 3 kb globulin-1 promoter was used to make constructs with one copy of E1 or CBHI. An exact comparison with the previous work using the 1.4 kb promoter was not possible as the genetic backgrounds are different and there was only a limited amount of T1 generation seed available for the 1.4 kb globulin-1 promoter construct. Nevertheless, the levels of E1 and CBHI between the two sets of promoters were compared but they did not show a statistically significant difference (not shown).

Plants transformed with one copy of E1 (CEA) or CBHI (BCU) under control of the 3 kb globulin-1 promoter were compared with plants with two copies of E1 (CEB) or CBHI (CCA) under control of the same promoter (FIG. 7). It is well established that there can be significant variation in protein accumulation between independent transformation events and even between plants from the same transformation event. There is also substantial precedent that a backcross program selecting the best expressing plants to carry forward at each generation results in a substantial increase in overall expression over time (Hood 2011). Thus, in addition to the overall mean accumulation, a comparison of a selection of the highest expressing plants or seeds may actually be a better indication of the best potential expression for a given construct. The ten highest expressing seeds for each construct were compared as well as the overall mean of all positive seeds. The mean level of E1 increased with the addition of a second copy of the plant transcription unit (PTU) by 50% and the mean value in the ten highest seeds showed a 2.4-fold increase in levels between constructs with one and two PTUs (FIG. 7A; CEA vs. CEB). No difference in the mean levels of all positive CBHI seed upon addition of a second copy was observed but a 1.8-fold increase was observed when comparing the mean of the ten seed with the highest enzyme activity (FIG. 7B; BCU vs. CCA).

To determine if additional copies of the promoter-cellulase transcription unit resulted in still higher expression, constructs with three or four copies were tested with the E1 and CBHI genes. For CBHI the average enzymatic activity in the three-copy construct (CCF) was 2-fold higher than that seen with two copies. However, for E1 the expression levels in the four-copy construct (CEC) are actually less than for the two-copy construct and only 1.2-fold higher than for the one copy construct (FIG. 7).

Rather than using the same promoter to confer expression in multiple PTUs, constructs with three copies of the E1 or CBHI cellulase genes under the control of three different promoters were tested (CEK and CCG). Each of these promoters has been shown to support expression of the GUS reporter gene at levels similar to or greater than the 1.4 kb globulin-1 promoter (Streatfield, 2010). When the three different promoters were used for CBHI (CCG), average expression levels were increased 1.8 fold relative to those observed with one PTU using the 3 kb globulin-1 promoter (BCU). The E1 construct with three different promoters (CEK) also showed 1.8 fold higher levels of expression compared to that of a construct with one PTU driven by the 3 kb globulin-1 promoter (CEA). When the ten highest seeds in the CBHI and EI groups are compared, both enzymes show a three-fold increase over the construct with one copy using the 3 kb globulin-1 promoter (BCU and CEA)(FIG. 7).

The E1 enzyme is targeted to the vacuole for all of the above constructs. Based on previous work (Hood, 2007), E1 targeted to the endoplasmic reticulum (ER) also resulted in high levels of activity. Therefore, a construct with one copy of E1 targeted to the vacuole and one copy of E1 targeted to the ER was evaluated to see if this could result in higher expression (construct CEJ). Expression for this construct was similar to that for the construct with two copies of E1 both targeted to the vacuole (FIG. 7A; CEB vs. CEJ). Finally a construct with two copies of E1 under control of the pr36 promoter as another example of multiple transcription units was examined. This construct also supported accumulation of E1 similar to the construct with two copies of the 3 kb globulin-1 promoter.

To further assess the differences in expression levels for the different constructs, statistical analysis was performed (FIG. 7). For both E1 and CBHI the mean expression levels for all positive seed in different constructs were not found to be statistically different. However, a clear trend towards increasing the mean activity was seen upon addition of multiple copies of the transcription units. When the seed demonstrating the ten highest levels of activity were compared for E1, statistically significant increases in accumulation were achieved with constructs containing multiple copies of the enzyme coding region (p<0.0001; FIG. 7). The groups of constructs as determined by the Tukey-Kramer test for E1 showed that CEK, CEB, and CEL had similar levels of activity and that it was higher than the other three constructs, although CEL and CEB could not be distinguished from CEJ. When seed demonstrating the ten highest levels of activity were compared for CBHI, statistically significant (p<0.0001) increases in accumulation were found, with BCU<CCA<CCF and CCG.

To establish the absolute amount of enzymatic activity in seed extracts, 4-methylumbelliferyl-β-D-cellobioside (MUC) assays were performed using known amounts of purified E1 and CBHI as standards. Due to limited availability of purified E1 and CBHI enzymes however, this was impractical for high-throughput assays. Instead, a commercial preparation of Trichoderma reesei was used to generate a standard curve for all tests with seed extracts. This was compared to the standard using the purified E1 and CBHI that could then be used to calculate activity. Total soluble protein (TSP) was also measured from T₁ seed from plants transformed with E1 and CBHI and used to further normalize inconsistencies in extraction between different samples. Constructs containing three PTUs under control of three different promoters (CEK and CCG) were then compared to pooled seed from maize lines harboring the constructs with E1 and CBHI under control of the 1.4 kb globulin1 promoter after multiple generations of optimization (Hood, 2011). The absolute amounts of cellulase in extracts was then determined by comparing the fluorescence units resulting from the assay of representative seed extracts to that of purified standards and was expressed as % TSP. The % TSP for E1 was calculated at 18.8% for the construct with three different promoters (CEK) and 7.1% for the 1.4 kb globulin-1-E1 extract (BCH). For CBHI the % TSP was 4.1% for the construct with three different promoters (CCG) and 3.2% for the 1.4 kb globulin1-CBH1 extract (BCC). However, it should be noted that the BCH and BCC samples are from pooled heterozygous seed and the expression levels in positive seed are likely to be two-fold higher.

To confirm the amount of cellulase as determined by MUC assay, extracts were also assayed by Western blots. The amount of total protein loaded on the gel was determined by the bicinchonic acid (BCA) protein assay and the amount of E1 or CBHI was calculated by using purified standards with the MUC assay. Increasing amounts of the purified enzymes resulted in a reasonably linear increase in signal (FIGS. 8A and 8B, lanes 7, 8, and 9). A good correlation is observed between the relative band intensities of cellulase on the blots and the amount of cellulase as determined by the MUC assay. These results confirm high levels of E1 and CBHI accumulation using these new constructs in transgenic maize and that most, if not all, of the enzyme is active. They also show that the transgenic protein levels in T1 seed using these new constructs are similar to that achieved after generations of optimization with the BCC and BCH constructs.

As transformation may result in integration into one or more locations, it is important to confirm that increases in expression are not simply due to multiple integrations in the chromosomes. Multiple integrations complicate traditional plant breeding approaches and make it difficult to sustain the high levels that can be seen in the T₁ seed. To obtain an estimate of copy number and integration patterns, Southern blot (Souther, 1975) analysis was carried out on genomic DNA preparations from selected lines. Blots from representative independent transformation events from the two best expressing constructs with three copies of CBHI under the control of the globulin1 promoter (CCF) and three copies of E1 under the control of different promoters (CEK) are shown in FIG. 9A. For both constructs, the lower band of 4.4 kb-5 kb is an internal fragment expected to be present within the construct. The presence of one additional higher molecular weight band resulting from hybridization to the last PTU (FIG. 9B) is consistent with a single insertion for at least some of the highest-expressing events.

The most promising plants from the T1 analysis were chosen to confirm that expression is maintained in the T₂ generation and to begin the process of optimization in germplasm. Seed from two plants for E1 and two plants for CBHI were planted in the greenhouse and back-crossed to an elite line (16038). Twenty-five T₂ seed from the resulting plants were ground into a powder and were assayed as described above. In this case only half of the pooled seed are expected to contain the transgene and therefore it is expected that the level of activity should be half of that obtained when comparing to the parent T₁ seed. The results shown in FIG. 10 indicate that the T₂ seed for constructs CEK, CEB, CCF, and CCG have maintained activity at least as high as the average activity in T1 seed for that construct and nearly as high as the average activity observed in T₁ seed from the corresponding event only. This provides evidence that the levels of activity seen in T₁ seed can be maintained or even increased as has been done previously for later generations (Hood, 2011).

Experimental Procedures Construct Preparation

A 3 kb fragment of the promoter region of the maize globulin-1 gene (Streatfield, 2010) was fused to the coding region for the T. reesei CBH I gene by standard molecular biology techniques (construct BCU). To prepare a construct with one copy of the Acidothermus cellulyticus E1 coding region downstream of 3 kb globulin-1 promoter (construct CEA), an NcoI-NheI fragment containing the E1 coding region was isolated from the previously described construct with E1 downstream of the 1.4 kb globulin-1 promoter (BCH) (Hood, 2007) and ligated to an NcoI/NheI fragment from BCU containing the vector and promoter sequence. The E1 (here SEQ ID NO: 18, see FIG. 14) and CBHI sequence (here SEQ ID NO: 19, see FIG. 15) used is that described at US Patent Application No. 20060026715, incorporated herein by reference in its entirety (the application showing E1 as sequence 1 and CBHI as sequence 4). CBHI sequence used is that described at GenBank accession No. L22656 and maize optimized The gene was maize optimized for the first 40 amino acids using a PCR based mutagenesis approach—this includes the 24 amino acid BAASS sequence. Codons D346 and D386 were also maize codon optimized to remove the potentially destabilizing sequences at those positions.

Based on previous studies (Hood, 2007) of variation in expression depending on subcellular localization, CBH1 constructs include the barley alpha amylase signal sequence (Rogers, 1985 #462) for cell wall localization and E1 constructs contain a vacuole targeting sequence (Holwerda, 1992 #935):

(SEQ ID NO: 15) Atggcccacgcccgcgtcctcctcctggcgctcgccgtcctggccacg gccgccgtcgccgtcgcctcctcctcctccttcgccgactccaacccg atccggccggtcaccgaccgcgccgcgtccacc All constructs were built using the shuttle vectors pGEM (Promega) or pCR2.1 TOPO® (Invitrogen) and transferred into the plant transformation vector pSB1 (Komari, 1996).

To prepare a construct with two copies of E1 under control of the 3 kb globulin-1 promoter the entire expression cassette was amplified by PCR using the single copy construct (CEA) as a template with the addition of Nhe1 restriction sites on both ends. This second copy was inserted into the NheI restriction site of the single copy construct to prepare construct CEB. To add a third and fourth copy (CEC), the expression cassette was amplified by PCR and with the addition of Nan or XbaI restriction sites on the ends. This expression cassette was inserted into restriction sites engineered into the two or three copy intermediate PCR primers. PCR products were sequenced and mutations were corrected by restriction fragment exchange with the wild type sequence by standard cloning techniques.

Preparation of expression cassettes for the second and third copies of CBHI (constructs CCA and CCF) was also accomplished by PCR amplification. To facilitate cloning of large DNA fragments the cassettes were amplified in two pieces roughly corresponding to the promoter and coding region. A SacII site in the extended globulin promoter and SacII sites in the pGEM vector multiple cloning site were used to reconstruct the full expression cassette in the pGEM shuttle vector before addition to the plant transformation construct in the pSB1 vector. For the second and third copies, AgeI and NheI restriction sites were engineered into the outer ends of the cassette respectively.

As a first step in preparation of constructs with cellulase enzymes under control of different promoters, intermediates were prepared in which the GUS coding region of earlier constructs (Streatfield, 2010) was replaced with CBHI or E1 coding sequence. For the globulin-2 and pr26 promoters, the promoter sequence was amplified by PCR. In some cases the sequence around the translation start site was engineered to correspond more closely to the optimal Kozak sequence. PCR products were sequenced and mutations were corrected by restriction fragment exchange with the wild type sequence by standard cloning techniques. The promoter was then inserted into the 3 kb globulin-1-CBHI plant transformation construct as an AgeI-NcoI fragment. This cloning was accomplished in multiple steps to accommodate an NcoI site in the CBHI coding region and an AgeI site in the globulin-2 promoter. The CBHI coding region was subsequently replaced with an NcoI-PacI fragment containing the E1 coding region to prepare the corresponding E1 construct.

For the pr36 promoter, the use of an NcoI site at the junction of the promoter and coding region was precluded by the presence of NcoI sites in both the promoter and the CBHI coding region. Thus the promoter and CBHI and E1 coding regions were amplified by PCR separately to engineer an XmaI site at the junction between the promoter and coding regions. The full expression cassettes were combined as two XmaI/SacII fragments in the pGEM vector.

For preparation of the construct with E1 under control of three different promoters (CEK) each new expression cassette was isolated as an AscI-MluI restriction fragment and ligated into the AscI restriction site of the preceding intermediate starting with the pr26-E1 expression cassette in pSB1. For preparation of the construct with CBHI under control of three different promoters (CCG), an expression cassette with CBHI under control of the pr36 promoter was added to the globulin-1-CBH1 construct as a PmeI-NotI fragment, and an expression cassette with CBH1 under control of the globulin-2 promoter was added to the resulting intermediate as an AscI fragment.

For preparation of a construct with E1 targeted to the vacuole and ER (CEJ), a KDEL sequence was added to the carboxy terminal end of the E1 coding region by PCR. The expression cassette was added to a construct with one copy targeted to the vacuole (CEA) as an AscI-MluI fragment.

Maize Transformation

Maize transformation was carried out as previously described using a method modified from Ishida, et al (Ishida, 1996). In brief, the constructs were transferred into the LBA4404 Agrobacterium strain containing the vector pSB1 (Komari, 1996) by a triparental mating procedure. The cointegrate DNA was then electroporated into Agrobacterium tumefaciens strain EHA101 (Hood, 1986). Maize embryos at roughly 2-4 mm were mixed with A. tumefaciens EHA101 with the appropriate vector for transformation. Plants were grown to maturity in the greenhouse and pollinated with HiII to produce T₁ seed.

MUC Assays

Six T1 seed were individually pulverized in 50 mM sodium acetate buffer pH 5.0 and total protein was determined by Bradford (Sigma or Bio-Rad) or BCA (Pierce) assay. Approximately 0.5-1.0 μg total soluble protein (TSP) was mixed with the substrate 4 methylumbelliferyl-beta-D-cellobioside substrate (#M6018, Sigma, St. Louis, Mo.) and incubated for 30 minutes for E1 or 2 hours for CBHI. Fluorescence was read at 360 and 460 nm. Samples were compared to a cellulase mixture from T. reesei as a standard (#C8546 Sigma, St. Louis, Mo.).

Typically, to evaluate a maize line overexpressing a protein of interest, the protein can be compared to a purified protein standard. In the case of the E1 and CBHI cellulase enzymes, however, accurate quantitation of large numbers of samples is complicated by a limited availability of purified enzymes for comparison in high-throughput assays. Therefore, for screening the large numbers of plants necessary to identify high-expressing lines, a relative measure of the cellulase activity of extracts based on the raw fluorescence units per microgram total soluble protein normalized to the Trichoderma reesei positive control was used. The average for all positive seed or for the highest-expressing ten seed was then calculated.

Statistical Analysis E1

An analysis of variance was carried out using the mixed procedure in SAS® v9.1 fitting a conditional hierarchical linear model. Each expression construct was considered a fixed effect. Events nested within constructs, and plants nested within events were considered random effects. Since the variances of the responses tended to increase as the construct means increased, constructs were placed into two groupings such that constructs within groups had approximately equal standard deviations and constructs between groups had unequal standard deviations. Specifically, constructs CEA (s.d.=36.501), and CEC (s.d.=47.183) were placed into one group and constructs CEB (s.d.=83.478), CEJ (s.d.=61.106) and CEK (s.d.=114.594) were placed into another. Significant differences were found among the construct means (p=0.0066). Post-hoc pairwise comparisons of construct means within these groups were carried out using the Tukey-Kramer method, allowing for equal standard deviations. Post-hoc pairwise comparisons of construct means between these groups were done using the Tukey-Kramer method, allowing for unequal standard deviations. The experiment-wise error rate was controlled at 0.05.

CBHI

The analysis of variance was carried out using the mixed procedure in SAS® v9.1 fitting a conditional hierarchical linear model. Each expression construct was considered a fixed effect. Events nested within constructs, and plants nested within events were considered random effects. There were no significant differences among the construct means (p=0.1251). Post-hoc pairwise comparisons of construct means are not appropriate.

Western Blot

Extracts were prepared from single seeds in 50 mM sodium acetate pH 5.0 buffer and assayed for total protein using the BCA assay and for cellulase activity using the MUC substrate. Varying amounts of protein were run on Bis-Tris 4-12% NuPAGE gels (Invitrogen, Carlsbad, Calif.). Protein was transferred to a PVDF membrane using the iBlot (Invitrogen, Carlsbad, Calif.). E1 and CBHI were purified as previously described (Hood, 2011). Antibodies raised in rabbits against bacterially expressed E1 and CBHI were used (Hood, 2011). Protein was detected with BCIP/NBT reagent (Sigma, St. Louis, Mo.).

DNA Blot Hybridization of Transgenic Lines

Genomic DNA was isolated from young leaf tissue using the Plant DNAEasy Maxi kit (Qiagen, Valencia, Calif.). Ten μg genomic DNA was digested with AgeI, NcoI, or NheI and run on a 1% agarose gel. DNA was transferred to Invitrogen Bright Star membrane by a standard capillary method in 0.4M NaOH, 1M NaCl alkaline transfer buffer. An approximately 1 kb fragment of E1 or CBHI was amplified by PCR and labeled as probe using the Bright Star psoralen-biotin kit (Invitrogen) as per the manufacturer's directions. The blots were hybridized using the probe at ca. 0.1 nM in 100 mM sodium phosphate pH 7.0, 6.7×SSC, 1% SDS, 0.065% bovine serum albumin, 0.065% polyvinylpyrolidone, 0.065% Ficoll, and 0.1 mg/mL salmon sperm DNA. The blots were washed twice for 20 minutes in 2×SSC, 0.4% SDS, twice for 20 minutes in 1×SSC, 0.25% SDS, and twice for 15 minutes in 0.5×SSC, 0.25% SDS. Signal was developed with the Bright Star BioDetect kit (Invitrogen) as per manufacturer's directions.

Discussion

A number of studies have attempted to express enzymes required for cell wall deconstruction in plants to enhance the use of agricultural wastes for biofuel production. Expression of E1 up to 26% total soluble protein (TSP) has been reported in the model species Arabidopsis, but levels have generally been lower in economically important crop plants (Austin-Phillips, 1999; Dai, 2000; Ziegler, 2000). For example, E1 has been expressed in transgenic maize at levels of 1-2% TSP (Biswas, 2006; Mei, 2009; Ransom, 2007) and at 4.9% TSP in rice (Oraby, 2007). When expression of enzymes including β-glucosidase, endoglucanase, exoglucanase, and xyloglucanse was targeted to the tobacco chloroplast, levels of up to 12% TSP were reported (Gray, 2009; Gray, 2011; Petersen, 2011). Expression of two xylanase enzymes under control of two different promoters, constitutive and grain-specific, has been achieved in maize, although expression resulted in unhealthy plants (Brunecky, 2011; Gray, 2011). E1 expression under control of a constitutive promoter in transgenic maize and tobacco permitted less pretreatment to achieve digestion comparable to the non-transgenic plants (Brunecky, 2011). Despite these advances, further improvements in levels and tissue-specific control of expression are required for economical biofuel production, especially for CBHI.

Future advances may require transformation of multiple transcription units, either for high-level expression of the same gene or expression of multiple genes. The use of different promoters for each transcription unit may alleviate gene silencing, reduce the chances of recombination, or prevent competition between two promoters for the same transcription factors. A set of recently characterized maize embryo-preferred promoters were shown to increase expression of the GUS gene over the well-established 1.4 kb version of the globulin1 promoter (Streatfield, 2010). However, the level of expression of GUS is relatively low and targeted to the cytoplasm. In this study, the functionality of these promoters was tested with CBHI and E1, two highly accumulated, secreted proteins in plants.

CBHI or E1 accumulation was increased by additional copies of the PTU containing the enzyme coding region. For both E1 and CBHI the addition of a second PTU with the extended globulin promoter resulted in an increase in transgenic protein accumulation when the ten highest seeds were analyzed. The addition of a third copy increased expression even more with CBHI, but for E1 cellulase expression decreased with a four-copy construct. There are a number of possible explanations for this but the most likely is gene silencing for the E1 construct, but not CBHI. For CBHI, a construct with three different promoters showed similar high expression to the construct with three PTUs under control of the same promoter. A construct with E1 under control of three different promoters showed elevated expression relative to all other constructs. This indicates that the relatively low amount of E1 in plants using the construct with four copies under control of the same promoter is not due to the inherent properties of the protein but possibly some form of gene silencing or recombination and that the use of different promoters may alleviate this obstacle. In general, the use of constructs with multiple PTUs increased expression by 2 to 3-fold for E1 and CBHI relative to constructs with only one PTU under control of the 3 kb globulin1 promoter when the highest ten seed are considered.

To confirm these results, Western blot analysis was performed as an alternative method of comparing the level of expression of the new constructs with earlier lines. The level of E1 and CBHI in crude extracts as determined by Western blots agreed with that obtained using the enzymatic assay. Expectations are that selection and backcrossing of these new constructs into elite germplasm, preparation of homozygous lines, and eventually homozygous hybrids will further increase expression significantly as has been shown for other proteins including cellulase (Hood, 2011). Although it is not uncommon to observe some decrease in expression in the first generation of crossing into elite germplasm, T₂ seed maintained enzyme activity levels as high or higher than the average expression levels in T₁ seed for the corresponding construct.

These results show increasing levels of expression can be achieved with multiple copies of a plant transcription unit for cellulase enzymes. They also confirm the ability of several embryo-specific promoters previously tested with a reporter gene to support expression of proteins of economic value. Future overexpression strategies may require the expression of multiple genes simultaneously (reviewed in Douglas, 2009; Halpin, 2005). A number of studies have used gene stacking, in particular for expression of genes involved in vitamin synthesis (Aluru, 2008; Naqvi, 2009; Naqvi, 2010). Others have attempted to increase expression of foreign proteins using multiple copies of the same gene under control of different promoters (Hennegan, 2005). However, many of these reports have used some form of co-transformation or traditional plant breeding. The availability of a variety of promoters for transgene expression that can be used within the same vector as described here provides more effective approaches.

These transgenic lines show great potential for use as a low cost source of cellulase that can reduce the need for expensive enzymes produced by fungal fermentation (Howard, 2007; Howard, 2011). The current data show expression at approximately 2.1 g E1 per kg seed and 0.62 g CBHI per kg seed in constructs with multiple PTUs under control of different promoters. Routine milling and de-germing of corn grain can increase the concentration in the germ ˜7-fold. It has been estimated that expression levels of 4% dry weight in the germ may be necessary to impact economical production of biofuels (Howard, 2011). Based on the data above, cellulase expression is now in this targeted range.

Example 2

The foregoing experiment was repeated in which two PTUs were constructed with the same promoter driving a heterologous nucleic acid molecule in a plant. In this instance, the heterologous nucleic acid molecules were hepatitis B antigen and aprotinin.

The Hepatitis B surface antigen (HBsAg) sequence, identical to the surface antigen protein sequence available in GenBank accession 562754.1 (adr subtype, small form i.e. S open reading frame without pre-S1 or pre-S2 sequences), was engineered to be codon optimized for expression in maize in all of the above constructs (See FIG. 11) and the barley alpha amylase signal sequence included in the vector. Rogers, J. C. (1985) “Two barley alpha-amylase gene families are regulated differently in aleurone cells” J. Biol. Chem. 260: 3731-3738. See FIG. 11 where the optimized hepatits B surface antigen nucleotide sequence used is shown (SEQ ID NO: 16), with the BAASS sequence in italics (SEQ ID NO: 17) and the ATG start codon and stop codon in bold. The promoter used was driven by the extended globulin-1 promoter (SEQ ID NO: 5). The plasmid map of the vector used is shown in FIG. 12. The aprotinin coding sequence is that disclosed at disclosed at U.S. Pat. No. 5,824,870, incorporated herein by reference in its entirety. The plasmid map of the vector used is shown in FIG. 13.

Maize plant cells were transformed as described in Experiment 1 above and production of the heterologous protein molecule determined. See, e.g. Hayden et al, “Production of highly concentrated, heat-stable hepatitis B surface antigen in maize” Plant Biotechnology Journal 2012 October; 10(8):979-84 and Zhong G., Peterson D., Delaney D., Bailey M., Witcher D., Register Iii J., Bond D., Li C., Marshall L., Kulisek E. (1999) Commercial production of aprotinin in transgenic maize seeds. Molecular Breeding 5:345-356. Results are summarized in the table below.

TABLE 5 Protein Production Aprotinin 1-copy 125 ug/g Aprotinin 2 copy 200 ug/g Hep B 1-copy 0.17% tsp Hep B 2 copy 0.27% tsp

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1. A method of increasing expression of a nucleic acid molecule, the method comprising introducing into a plant or plant part at least two repeats of a plant transcription unit (PTU), said PTU comprising a promoter operably linked to a nucleic acid molecule encoding a product, wherein said product encoded by said nucleic acid molecule is increased compared to a plant or plant part comprising a single PTU.
 2. The method of claim 1, wherein said product encoded is chosen from endo-β-1,4-glucanase (E1), exo-β-1,4-glucanase (CBH1), hepatitis B and aprotinin.
 3. The method of claim 1, wherein said product encoded comprises a cellulase enzyme.
 4. The method of claim 1, wherein said product encoded is chosen from endo-β-1,4-glucanase (E1) and exo-β-1,4-glucanase (CBH1).
 5. The method of claim 1, wherein said promoter comprises an embryo-preferred promoter.
 6. The method of claim 1, wherein said promoter comprises a globulin promoter.
 7. The method of claim 1, wherein said promoter is chosen from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 20 and SEQ ID NO:
 21. 8. The method of claim 1, comprising introducing into said plant three or four of said PTUs.
 9. The method of claim 1 wherein said plant cell is a corn plant cell.
 10. The method of claim 1, comprising introducing into said plant or plant part two of said PTUs, wherein said product expressed comprises endo-β-1,4-glucanase (E1).
 11. The method of claim 1, wherein said plant part comprises seed and wherein said product encoded comprises endo-β-1,4-glucanase (E1) and increasing production of said E1 by at least two fold in said seed compared to production of said E1 in said seed comprising a single PTU.
 12. The method of claim 1, comprising introducing into said plant or plant part three of said PTUs, wherein said product expressed comprises exo-β-1,4-glucanase (CBH1).
 13. The method of claim 1, said plant part comprises seed and wherein said product encoded comprises exo-β-1,4-glucanase (CBH1) and increasing production by at least 1.7 fold compared to production of said CBH1 in said seed comprising a single PTU.
 14. A method of increasing expression of a nucleic acid molecule, the method comprising introducing into a plant or plant part at least two plant transcription units (PTU) comprising a promoter operably linked to a nucleic acid molecule encoding a product, each promoter different from the other promoter, wherein the production of said enzyme is increased compared to a plant or plant part comprising a single promoter operably linked to a single nucleic acid molecule.
 15. The method of claim 14, comprising introducing or transforming a vector comprising said PTU into said plant or plant part.
 16. The method of claim 14 wherein said plant cell is a corn plant cell.
 17. The method of claim 14, wherein said PTU comprises components in addition to said promoter operably linked to said nucleic acid molecule and said nucleic acid molecule, and wherein said other components of said at least two PTUs are the same.
 18. The method of claim 14, wherein said product encoded comprises a cellulase enzyme.
 19. The method of claim 14, comprising introducing three PTUs into said plant cell, each PTU comprising three different promoters operably linked to a nucleic acid molecule encoding endo-β-1,4-glucanase (E1) or exo-β-1,4-glucanase (CBH1).
 20. The method of claim 14 further comprising producing a plant comprising seed from said plant cell and wherein said E1 is produced at a level of at least 18% total soluble protein in said seed.
 21. The method of claim 14, further comprising producing a plant comprising seed from said plant cell and wherein said CBH1 is produced at a level of at least 4% total soluble protein in said seed.
 22. The method of claim 14, wherein said three different promoters chosen from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 20 and SEQ ID NO:
 21. 